1. Field of the Invention
This invention relates generally to delivery of biomolecules using biodegradable polymer-peptides. More particularly, this invention relates to acid sensitive biodegradable polyacetal conjugating peptides complexed with polynucleotides, methods for making the complexes, and methods for using them in polynucleotide delivery applications.
2. Description of the Related Art
There is a need for non-viral drug delivery systems having desirable properties such as low immunogenicity, amenable to production on a relatively large scale, and which can be easily modified to provide a range of biological properties. See Mulligan, R. C., “The basic science of gene therapy,” Science 260, 926-932 (1993); and Luo, D. & Saltzman, W. M. “Synthetic DNA delivery systems,” Nat. Biotechnol. 18, 33-37 (2000). However, non-degradable cationic polymers such as poly(lysine) and polyethyleneimine (PEI) can have significant cytotoxicity. See Choksakulnimitr, S., Masuda, S., Tokuda, H., Takakura, Y. & Hashida, M., “In vitro cytotoxicity of macromolecules in different cell culture systems,” J. Control Release 34, 233-241 (1995); Brazeau, G. A., Attia, S., Poxon, S. & Hughes, J. A., “In Vitro Myotoxicity of Selected cationic macromolecules used in non-viral gene therapy,” Pharm. Res. 15, 680-684 (1998); and Ahn, C.-H., Chae, S. Y., Bae, Y. H. & Kim, S. W. “Biodegradable poly(ethylenimine) for plasmid DNA delivery,” J. Control. Release 80, 273-282 (2002).
To reduce cytotoxicity, some efforts have been made to develop degradable cationic polymers (polycations). See Ahn, C.-H., Chae, S. Y., Bae, Y. H. & Kim, S. W., “Biodegradable poly(ethylenimine) for plasmid DNA delivery,” J. Control. Release 80, 273-282 (2002); Lynn, D. M.; Anderson, D. G.; Putman, D.; Langer, R., “Accelerated Discovery of Synthetic Transfection Vectors: Parallel Synthesis and Screening of a Degradable Polymer Library,” J. Am. Chem. Soc. 123, 8155-8156 (2001); Lim, Y. et al., “Biodegradable Polyester, Poly[α-(4-Aminobutyl)-1-Glycolic Acid], as a Non-toxic Gene Carrier,” Pharmaceutical Research 17, 811-816 (2000); Lim, Y., Kim, S., Suh, H. & Park, J.-S., “Biodegradable, Endosome Disruptive, and Cationic Network-type Polymer as a High Efficient and Non-toxic Gene Delivery Carrier,” Bioconjugate Chem. 13, 952-957 (2002); Lim, Y. K., S.; Lee, Y.; Lee, W.; Yang, T.; Lee, M.; Suh, H.; Park, J., “Cationic Hyperbranched Poly(amino ester): A Novel Class of DNA Condensing Molecule with Cationic Surface, Biodegradable Three-Dimensional Structure, and Tertiary Amine Groups in the Interior,” J. Am. Chem. Soc. 123, 2460-2461 (2001); and Tuominen, J. et al., “Biodegradation of Lactic Acid Based Polymers under Controlled Composting Conditions and Evaluation of the Ecotoxicological Impact,” Biomacromolecules 3, 445-455 (2002). However, under physiological conditions, these cationic polymers are susceptible to degradation via base-catalyzed hydrolysis.
Acid-sensitive polymers containing acetal linkages has been reported, see Tomlinson, R. et al., “Pendent Chain Functionalized Polyacetals That Display pH-Dependent Degradation: A Platform for the Development of Novel Polymer Therapeutics,” Macromolecules 35, 473-480 (2002); and Murthy, N., Thng, Y. X., Schuck, S., Xu, M. C. & Fréchet, J. M. J., “A Novel Strategy for Encapsulation and Release of Proteins: Hydrogels and Microgels with Acid-Labile Acetal Cross-Linkers,” J. Am. Chem. Soc. 124, 12398-12399 (2002).
Using peptides for delivery of proteins has attracted a great deal of attention in the life science research area. Due to their natural properties, peptides were believed to be biocompatible. See Schwarze, S. R.; Ho, A.; Vocero-Akbani, A.; Dowdy, S. “In Vivo Protein Transduction: Delivery of a Biologically Active Protein into the Mouse.” Science 285, 1560-1572 (1999), and Tung, C.; Weissleder, R. “Arginine containing peptides as delivery vectors.” Adv. Drug Deliv. Rev. 55, 281-294 (2003). Recently, the utility of peptides as enhancers of gene delivery was reported. See Hawley-Nelson, P.; Lan, J.; Shih, P.; Jessee, J. A.; Schifferli, K. P.; Gebeyehu, G.; Ciccarone, V. C.; Evans, K. L. “Peptide-enhanced transfections.” US Patent Application, US20030069173A1 (2003), and Hawley-Nelson, P.; Lan, J.; Shih, P.; Jessee, J. A.; Schifferli, K. P.; Gebeyehu, G.; Ciccarone, V. C.; Evans, K. L. “Peptide-enhanced transfections.” US Patent Application, US20030144230A1 (2003). Peptide-mediated transfection also reported. See Legendre, J. Y.; Trzeiak, A.; Bohrmann, B.; Deuschle, U.; Kitas, E.; Supersaxo, A. “Dioleoylmelittin as a Novel Serum-Insensitive Reagent for Efficient Transfection of Mammalian Cells.” Bioconjugate Chem. 8, 57-63 (1997), Futaki, S.; Ohashi, W.; Suzuki, T.; Niwa, M.; Tanaka, S.; Ueda, K.; Harashima, H.; Sugiura, Y. “Stearylated Arginine-Rich Peptides: A New Class of Transfection Systems.” Bioconjugate Chem. 12, 1005-1011 (2001), Siprashvili, Z.; Scholl, F. A.; Oliver, S. F.; Adams, A.; Contag, C. H.; Wender, P. A.; Khavari, P. A. “Gene Transfer via Reversible Plasmid Condensation with Cysteine-Flanked, Internally Spaced Arginine-Rich Peptides.” Human Gene Ther. 14, 1225-1233 (2003), Haines, A. M.; Phillips, R. O.; Welsh, J. H.; Thatcher, D. R.; Irvine, A. S. “Compositions and Methods for highly Efficient Transfection.” PCT International Application, WO9835984 (1998), Haines, A. M.; Phillips, R. O.; Welsh, J. H.; Thatcher, D. R.; Irvine, A. S.; Craig, R. K. “Compositions and Methods for highly Efficient Transfection.” U.S. Pat. No. 6,479,464B1 (2002), Haines, A. M.; Phillips, R. O.; Welsh, J. H.; Thatcher, D. R.; Irvine, A. S.; Craig, R. K. “Compositions and Methods for highly Efficient Transfection.” US Patent Application, US20030100496 (2003), Divida, G.; Morris, M.; Mery, J.; Heitz, F.; Fernandez, J.; Archdeacon, J.; Horndorp, K. “Peptide-Mediated Delivery of Molecules into cells.” PCT International Application, WO0210201A2 (2002), Divida, G.; Morris, M.; Mery, J.; Heitz, F.; Fernandez, J.; Archdeacon, J.; Horndorp, K. “Peptide-Mediated Transfection Agents and Methods of Use.” US Patent Application, US20030119725A1 (2003), Wolff, J. A. “Compositions and Methods for Drug Delivery Using pH sensitive Molecules.” PCT International Application. WO0075164A1 (2000), Phillips, R. O.; Welsh, J. H.; Husain, R. D. “Membrane disruptive peptides covalently oligomerized.” PCT International Application. WO0064929 (2000), Legendre, J.; Supersaxo, A.; Trzeciak, A. “Peptide Conjugates for Transfecting Cells.” U.S. Pat. No. 6,030,602 (2000), Wadwha, M. S.; Rolland, A.; Logan, M.; Sparro, J. T. “Lipophilic and/or lytic peptides for specific delivery of nucleic acids to cells.” PCT International Application, WO9850078 (2000).
However, small peptides such as octaarginine without a tethered lipid chain are not particularly good for transfection. See Futaki, S.; Ohashi, W.; Suzuki, T.; Niwa, M.; Tanaka, S.; Ueda, K.; Harashima, H.; Sugiura, Y. “Stearylated Arginine-Rich Peptides: A New Class of Transfection Systems.” 12, 1005-1011 (2001), and Tung, C.; Weissleder, R. “Arginine containing peptides as delivery vectors.” Adv. Drug Deliv. Rev. 55, 281-294 (2003).